Influence of Protein Synthesis on NO₃⁻ Reduction, NH₄⁺ Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Abstract

Changes in the concentrations of NH4' and amides during the growth of suspension culures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only N03-as a nitrogen source, the concentrations of NHE44 and amies increased to 4.0 x 10-1 and 5.9 micromoles per gram fresh weight, respectively. The or as 'N03 + NH4+,' contained 1,920 pmol N03-or the same amount of N03-+ 72.8 ,mol NH4+, respectively. Cultures were initiated in both types of medium by inoculation with 0.5 g of cells from 14-day-old N03 + NH4+ cultures. Because of differences in the growth rates in the two media (17, 18) cells were grown for 14 days in the NO3 + NH4+ medium and for 21 days in the N03 medium. At regular intervals during the growth cycles, 1-to 15-g samples ofcells were harvested and homogenized in 80% ethanol with a Blackstone Ultrasonicator set at 80%Yo of full power for 2 min. The ethanol-soluble compounds were separated from insoluble material by filtration. The ethanol-soluble samples were then assayed for their NH4+ and amide contents by steam distillation and titration (2).Inhibitors. Ten-day-old cultures grown in NO3 medium were used for the inhibitor studies, because of their rapid rate of protein synthesis (17) and their low NH4' content (Table I). Preliminary studies were performed, as previously described (8), with either acetate-U-14C or leucine-U-4C to determine the effectiveness of various concentrations of cycloheximide and puromycin to inhibit protein synthesis of 10-day-old cells grown in N03 medium. Repeated experiments showed that 3.6 X 106 M cycloheximide and l0-4 M puromycin inhibited protein synthesis by approximately 85% and 40%, respectively.The effect of these inhibitors on the accumulation of NH4+ and amides by rose cells was determined by incubating a 25-g sample of 10-day-old cells in media containing either 3.6 x 10-M cycloheximide or l0-4 M puromycin. The incubations were carried out in sintered glass funnels containing 50 ml of medium which had been filtered from 10-day-old cultures. Air forced up through the funnel provided aeration and mixing. Following 0, 2, and 4 h ofincubation, cell aliquots were harvested, and the ethanol-soluble compounds were extracted as described in the previous section.The ethanol-soluble fractions were then subjected to steam distilwww.plantphysiol.org on March 25, 2019 -Published by Downloaded from

Nitrate and ammonium accumulation in bermudagrass in relation to nitrogen fertilization and season

“…No toxic ammonium concentrations were ever found in the different harvests if we consider that NH~-N concentrations in plant tissues lower than 1400~g.g-~DW were not toxic (Bradford and Fletcher, 1982). Our annual average concentrations did not surpass such values.…”

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

“…By 38 d, about one-third of the total nitrate still remains in the medium. Similar patterns of N uptake in cell cultures of tobacco and rose cells have been observed, including the preferential use of ammonium and the inability to totally deplete the available nitrate (2,4,6,15).…”

“…The total GOGAT activity units decreased by more than 25% during the last 2 weeks of culture. While the GDH enzyme forms found in leaves and cell cultures may not be involved in ammonium assimilation (4,14), these enzymes may still serve an important role in total N metabolism of the plant cell (13 (Fig. 2B).…”

Rosa spp. (Roses): In Vitro Culture, Micropropagation, and the Production of Secondary Products

“…However, Bradford and Fletcher (1982) have used this system to show that the early steps of nitrogen assimilation (nitrate reductase and nitrite reductase) are not regulated by a similar end-product control mechanism.…”